Evidence that atypical protein kinase C-lambda and atypical protein kinase C-zeta participate in Ras-mediated reorganization of the F-actin cytoskeleton.

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of the atypical aPKC isotypes lambda and zeta, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-lambda and aPKC-zeta, as well as antisense constructs encoding RNA-directed against isotype-specific 5' sequences of the corresponding mRNA, abrogates the Ha-Ras-induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-alpha was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-lambda and aPKC-zeta lead to a disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3'-kinase (PI3K) inhibitors wortmannin and LY294002 are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-lambda acts upstream of PI3K and Rac-1, whereas aPKC-zeta functions downstream of PI3K and Rac-1. This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-lambda or aPKC-zeta results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-zeta was abrogated by coexpression of DN Rac-1 N17.

Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides.

We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.

Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation.

The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.

Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes.

T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-O K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-enRasS17N completely inhibited in the PKC-O A148E-induced signal, identifying PKC-theta as a specific constituent upstream of or parallel to Ras in the signaling cascade leading to AP transcriptional activation.

Transcellular activation of the HTLV promoter by human hematopoietic cells.

We examined the ability of hematopoietic cells to transactivate the HTLV promoter by a transcellular mechanism. HeLa cells containing a CAT reporter gene driven by the HTLV-2 promoter were cocultivated with hematopoietic cells of the B-(Raji), T-(HuT78, Jurkat) and monocyte/promyelocytic (THP-1, U937 and HL60) lineages. Cocultivation with U937 and HuT78 cells constitutively and significantly transactivated the HTLV-2 promoter, while no effect was observed with the other lines. However, activation of other T-cell lines (CEM, Jurkat, Molt-3 and MT-4) with a combination of phorbolester and phytohemagglutinin also resulted in potent transactivation. Supernatant from HuT78 cells exhibited detectable transactivating activity, suggesting that the activation is mediated by a secreted factor(s). This factor also transactivates the HTLV-1 promoter. We used a panel of HTLV-1 LTR deletion mutants to map the responsive elements to this factor(s). Unlike the response element to the HTLV transactivator protein, Tax, which can be mapped to a small region in the enhancer, maximal transactivation by the cellular factor(s) required the complete U3 sequence. Transcellular activation of the HTLV promoter by activated T-cells may play a role in the development of leukemia in HTLV infected individuals.

Role of 7,8-dihydroneopterin in T-cell apoptosis and HTLV-1 transcription in vitro.

Adult T-cell leukemia is associated with high levels of neopterin, released in large amounts from human macrophages upon stimulation with interferon-gamma. Recent data suggested a potential role of neopterin-derivatives in oxygen radical-mediated processes, and evidence accumulates that oxidative stress is involved in the pathogenesis of viral diseases. We now report that increased concentrations of 7,8-dihydroneopterin may lead to enhanced apoptosis and disturbance of the redox-balance of human leukemic Jurkat T cells. Additionally, we demonstrate that 7,8-dihydroneopterin and hydrogen peroxide activate the type 1 human T-cell leukemia virus (HTLV-1) long terminal repeat (LTR). Furthermore, we found that the activity of the HTLV-1 transactivator protein Tax is amplified by an elevated concentration of 7,8-dihydroneopterin. Tax did not significantly augment 7,8-dihydroneopterin mediated apoptosis. Based on our data we propose that 7,8-dihydroneopterin may be involved in the progression to higher stages of HTLV-1 associated disease.

Inhibition of HIV-1 expression by HIV-2.

HIV-1 and HIV-2 are co-endemic in certain geographic areas. HIV-2 is more weakly pathogenic than HIV-1, and progression to AIDS occurs less frequently and over a longer period of time. Recent epidemiologic studies suggest that individuals infected with HIV-2 have a lower risk of HIV-1 infection. Both immune mechanisms and various modes of viral interference have been proposed to account for these results. Our findings, described in this paper, suggest that HIV-2 inhibits HIV-1 replication. To study the molecular interactions between HIV-1 and HIV-2, proviral clones were transfected alone or in combination into the human T cell line CEM. LTR-CAT indicator constructs were included for the purpose of monitoring viral promoter activity. Viral replication in transfected cells was monitored by p24 antigen capture assay of cell culture supernatants and Western blot analysis of cell extracts. HIV-2 inhibited HIV-1 replication as determined by intracellular and extracellular p24 antigen levels. Similar results were obtained with simultaneous virus infection using HIV-1 and HIV-2, rather than transfections of proviral DNA. Using cotransfection of HIV-1 and HIV-2 LTR indicator gene constructs, the mechanism of inhibition was found to be suppression of the HIV-1 LTR by HIV-2. The inhibitory effect of HIV-2 is not due to Tat-2, but appears to discriminate between the HIV-1 and HIV-2 LTRs based on differences in the Tat activation response element, TAR. These results suggest both a molecular mechanism for HIV-2 interference with HIV-1 replication and a potential molecular approach to therapy.

Construction and characterization of lck- and fyn-specific tRNA: ribozyme chimeras.

Two src-family protein tyrosine kinases (PTKs), p56lck, and p59fyn, are thought to play an important role in the antigen-specific T cell receptor (TCR)/CD3-initiated signaling pathway, but their relative contribution to these events is not clearly defined. Here, we have explored the potential of catalytic RNA molecules, or ribozymes, as tools for selectively inhibiting expression of the corresponding target genes in T cells. Several lck- or fyn-specific hammerhead ribozymes were synthesized, cloned into a bacterial transcription vector, and found to display specific catalytic activity in vitro. In order to achieve stable high-level ribozyme expression in intact cells, selected ribozymes were subsequently cloned into a retroviral vector (DC-T5T) immediately downstream of a tRNA(met) transcription unit. Upon retroviral transduction of a human leukemic T cell line (Jurkat), two out of four chimeric tRNA:ribozymes, fyn-1 and lck-1, were stably expressed at levels of approximately 10,000 or approximately 25,000 copies/cell, respectively. Ribozyme expression was associated with a reduction of up to 80% (lck) or 61% (fyn) in endogenous target mRNA by comparison to the corresponding transcript levels in control clones transfected with vector alone. By contrast, expression of the corresponding target proteins was not reduced, suggesting a post-transcriptional compensatory mechanism that increases translation or stability of the p56lck and/or p59fyn proteins.

Complement-mediated enhancement of HIV-1 infection of the monoblastoid cell line U937.

To assess the role of complement and complement receptors in HIV-1 infection of monocytes and macrophages, we studied the infectivity of HIV-1, isolated from the peripheral blood of a patient with subacute AIDS-related encephalopathy, on the human monoblastoid cell line U937. HIV-1 and HIV-1-infected cells were capable of activating the complement system via the classical and the alternative pathways, respectively. Low concentrations of HIV-1 were able to infect U937 cells more easily in the presence than in the absence of complement. At higher virus concentrations, infectivity was no longer facilitated by the presence of complement. Infection of U937 cells was reduced in the presence of any of the monoclonal antibodies (MAbs), OKT4a (anti-CD4), OKM1 (anti-CR3), or M522 (anti-CR3). A combination of all three of these MAbs reduced the infection by an even greater amount. These data indicate that complement receptors may be a port of entry for complement-coated HIV-1.

Effects of neopterin-derivatives on H2O2-induced luminol chemiluminescence: mechanistic aspects.

Neopterin, 6-D-erythro-1',2',3'-trihydroxypropyl-pterin, and its dihydroform, 7,8-dihydro-neopterin, are synthesized by human monocytes/macrophages upon stimulation by interferon-gamma. In the presence of iron chelator complexes neopterin enhances hydrogen peroxide-induced luminol chemiluminescence at neutral or slightly alkaline pH (7.5). In contrast, 7,8-dihydroneopterin scavenges chemiluminescence independently from the pH value and iron. In this study, we explored in more detail the mechanism possibly involved: analysis of the reaction products shows that 7,8-dihydroneopterin is oxidized and degraded to 7,8-dihydroxanthopterin and xanthopterin, whereas the neopterin molecule is not chemically altered during the chemiluminescence reaction. Investigations of the neopterin-induced effect show that mannitol, a scavenger of hydroxyl radicals, does not alter the enhancing effect of neopterin. L-histidine, which scavenges singlet oxygen almost as effective as hydroxyl radicals, reduces the enhancing effect of neopterin. However, singlet oxygen was not detectable during the reaction by measuring monomol light emission (1270 nm). When replacing hydrogen peroxide by 3-morpholinosydnonimine, a generator of hydroxyl radicals, or naphthalene-endoperoxide, a generator of singlet oxygen, in the luminol chemiluminescence assay, neopterin shows no enhancing effect irrespective of the presence of iron-(III)-EDTA. The data suggest that neopterin enhances hydrogen peroxide-induced luminol chemiluminescence in the presence of iron-(III)-EDTA by formation of a catalytic complex that seems to favor the formation of oxygen intermediates which derive from hydrogen peroxide and react with luminol.

Neopterin and 7,8-dihydroneopterin induce apoptosis in the rat alveolar epithelial cell line L2.

The neopterin derivatives, neopterin and 7,8-dihydroneopterin, modulate the cellular oxidant-antioxidant balance as well as the expression of the inducible nitric oxide synthase (iNOS) gene. Since apoptosis can be induced by reactive oxygen intermediates and nitric oxide (NO) we investigated whether these neopterin derivatives induce apoptotic cell death. As model we selected the rat alveolar epithelial cell line L2. 24 h incubation of neopterin (1-1000 microM) as well as 7,8-dihydroneopterin (1-1000 microM) resulted in a significant increase of percent apoptotic cells (measured by FACS analysis). Coincubation of both pteridines with the cytomix (interferon-gamma plus tumor necrosis factor-alpha) led to a significantly higher apoptosis than the cytomix alone. In contrast to the cytomix, no iNOS gene expression and no NO release could be detected after incubation with neopterin or 7,8-dihydroneopterin. We conclude that neopterin and 7,8-dihydroneopterin are per se inducers of apoptosis which is not mediated by nitric oxide. This may be of importance in inflammatory pulmonary diseases associated with an activation of the cellular immune system.

Effect of neopterin and 7,8-dihydroneopterin on tumor necrosis factor-alpha induced programmed cell death.

Tumor necrosis factor-alpha and the formation of reactive oxygen intermediates are central mediators of apoptosis. Recent data indicated a role of neopterin and 7,8-dihydroneopterin in oxygen radical mediated processes. We have therefore investigated the effect of neopterin-derivatives on TNF alpha induced apoptosis of the monocyte-like cell line U937. At an elevated concentration 7,8-dihydroneopterin was found to superinduce TNF alpha mediated programmed cell death due to the formation of reactive oxygen intermediates. Our results imply that in combination with TNF alpha high concentrations of 7,8-dihydroneopterin enhances apoptosis due to oxidative stress on cells.

An efficient expression, purification and immunodetection system for recombinant gene products.

We describe a modification of the mammalian expression vector pRc/CMV, which drives expression of inserted genes from either the human cytomegalovirus (CMV) immediate-early promoter or the bacteriophage T7 RNA polymerase promoter. The modification is designed to allow expression, simple purification and specific immunodetection of recombinant fusion proteins. The modified plasmid, termed pTag/CMV-neo, encodes a Kozak consensus ribosome-binding site (RBS) and a 30-amino acid fusion tag peptide. This peptide consists of a metal ion-binding site, (His)6, for single-step affinity purification using Ni(2+)-chelating resin and a multi-purpose HIV-1-derived peptide (p18HIV). This viral epitope can be used to identify, detect and characterize target fusion proteins in conjunction with a specific monoclonal antibody H902 that does not display cross-reactivity with cellular proteins. The H902 production hybridoma cell line is reagent #521 from the NIH AIDS Research and Reference Program.

HIV and HIV-infected cells differentially activate the human complement system independent of antibody.

The human retroviruses HTLV-I and HIV-I have previously been shown not to be lysed by human serum. An interaction between HIV and the complement system, however, has not been investigated in any detail. In this report we show that purified HIV as well as HIV-infected cells activate the complement system. In the case of virus-infected cells this activation is mediated by the alternative pathway of complement, whereas the classical pathway seems to be in operation for the triggering of the complement system by purified virus and recombinant envelope glycoprotein (gp 160). We demonstrate that this leads to the deposition of C3b and/or C3bi on the surface of infected cells. But the HIV-infected cells are not lysed by human complement. C3 fragments deposited on the surface of HIV-infected cells are capable of mediating immune adherence to complement receptor-bearing cells, such as human erythrocytes and phagocytes. Whether this might have an influence on infectivity of HIV for certain cells bearing complement receptors has yet to be shown.

Further evaluation of soluble CD4 as an anti-HIV type 1 gene therapy: demonstration of protection of primary human peripheral blood lymphocytes from infection by HIV type 1.

We previously reported on the construction of retroviral vectors that produce a secreted form of the HIV-1 receptor, T cell antigen CD4 (Morgan et al., AIDS Res Hum Retroviruses 1990;6:183-191). In this article we test the ability of these sCD4-expressing retroviral vectors to protect human T-cell lines or primary T cells from HIV-1 infection. To demonstrate that protection from HIV-1 infection is mediated by the soluble nature of this protein, two coculture protection experiments were conducted. In these experiments, sCD4-expressing retroviral vectors were used to engineer mouse NIH 3T3 cells. In one coculture experiment the human SupT1 cell line was added directly to the culture of sCD4-producing NIH 3T3 cells, and in another experiment the two cell types were separated physically by a semipermeable membrane. In both coculture configurations, the T cell line was protected from HIV-1 challenge as measured by syncytium formation and indirect immunofluorescent assays. In addition, the SupT1 line was directly engineered with sCD4-expressing retroviral vectors and shown to be protected from HIV-1 challenge. As a prelude to further preclinical studies, we tested the ability of retroviral vectors to transduce primary human peripheral blood lymphocytes (PBLs). Conditions used to stimulate T cell growth resulted in significant shifts in the CD4/CD8 cell in favor of CD8 cells. Retroviral-mediated gene transfer under these conditions resulted in low levels of gene transfer (< 5%).(ABSTRACT TRUNCATED AT 250 WORDS)

trans-Activation of the HIV type 1 promoter by 7,8-dihydroneopterin in vitro.

Infection with human immunodeficiency virus type 1 and 2 is associated with elevated concentrations of neopterin, released in large quantities by human macrophages on stimulation with interferon gamma. Evidence has suggested a potential role of neopterin derivatives in oxygen radical-mediated processes. Here we show that the redox-sensitive transcription factors AP-1 and NF-kappaB are activated by 7,8-dihydroneopterin, either directly (AP-1), or by the synergistic action with tumor necrosis factor alpha (NF-kappaB). We could further demonstrate that 7,8-dihydroneopterin enhances HIV-1 expression as shown in transient transfection assays using HIV-1 CAT promoter-reporter gene constructs. In sera of HIV+ patients 7,8-dihydroneopterin significantly correlated with neopterin and HIV-1 p24 antigen. On the basis of our data we therefore assume that 7,8-dihydroneopterin might augment progression to higher stages of HIV-associated disease.

Chronic immune stimulation, oxidative stress, and apoptosis in HIV infection.

Infection with the human immunodeficiency virus (HIV) is accompanied by a decrease in CD4+ T cell numbers and the ultimate disruption of immunological functions. In sera of infected patients, elevated levels of interferon-gamma are detected, which is indicative of an activated TH1-type immune response. T-cell-derived interferon-gamma leads to the expression of various proinflammatory cytokines and enhanced macrophage capacity to secrete reactive oxygen intermediates. In addition, interferon-gamma is the major stimulator for the biosynthesis of neopterin and its reduced form, 7,8-dihydroneopterin. Neopterin is known as a sensitive immune activation marker in clinical laboratory diagnosis. Recent data implied a potential role of neopterin derivatives in oxygen free-radical-mediated processes, e.g. high concentrations of 7,8-dihydroneopterin were found to interfere with the oxidant-antioxidant balance, and may lead to apoptosis of human cells. In addition, 7,8-dihydroneopterin was found to be effective in the activation of redox-sensitive transcription factors and in the induction of HIV-1 gene expression. In this commentary, we describe our current view as to how neopterin derivatives, in concert with cytokines and reactive oxygen intermediates, may lead the way to the final destruction of the cellular immune system.

7,8-Dihydroneopterin-induced apoptosis in Jurkat T lymphocytes: a comparison with anti-Fas- and hydrogen peroxide-mediated cell death.

Activated cell-mediated immunity, associated for example with HIV infection, is accompanied by elevated concentrations of neopterin and 7,8-dihydroneopterin. Recent data have indicated a role of neopterin derivatives in virus activation and apoptotic cell death, processes likely to involve the action of oxygen free radicals. Because T cell death in AIDS is likely to involve the Fas/Fas ligand system and the action of oxygen free radicals and 7,8-dihydroneopterin, we compared the kinetics and sensitivity of apoptotic cell death of human leukemic Jurkat T cells to that of treatments with 7,8-dihydroneopterin, anti-Fas, and H2O2. Upon incubation with 5 mM 7,8-dihydroneopterin and 50 microM hydrogen peroxide over a period of 24 hr, bimodal kinetics were observed with peaks at 5.5 hr (7,8-dihydroneopterin, 13.1%; H2O2, 11.4%) and at 24 hr (7,8-dihydroneopterin, 11.2%; H2O2, 13.2%). In contrast, anti-Fas (20 ng/mL)-induced apoptosis increased steadily over time, peaking at 11 hr (43.2%). Interestingly, anti-Fas-induced apoptosis was suppressed upon co-incubation with 7,8-dihydroneopterin and H2O2 by 62% and 68%, respectively. We also compared the sensitivity to drug treatments of apoptosis induced by 7,8-dihydroneopterin, anti-Fas antibodies, and H2O2. 7,8-Dihydroneopterin-mediated, and similarly anti-Fas- and H2O2-mediated, apoptosis was not inhibited by a broad range of pharmacological inhibitors, such as actinomycin D, cycloheximide, cyclosporin A, and various protein kinase inhibitors. On the contrary, inhibitors with antioxidant abilities, such as pyrrolidinedithiocarbamate, significantly blocked 7,8-dihydroneopterin-, H2O2- as well as anti-Fas-mediated apoptosis. These results imply that 7,8-dihydroneopterin-, H2O2-, and anti-Fas-mediated cell death might involve related redox sensitive signal transduction pathways.

7,8-Dihydroneopterin upregulates interferon-gamma promoter in T cells.

Activated cell-mediated immunity is accompanied by elevated concentrations of interferon-gamma leading to the secretion of neopterin-derivatives which are known as sensitive immune activation markers in clinical laboratory diagnosis. Recent data imply a potential role of neopterin-derivatives in oxygen-free radical-mediated processes and a direct impact of 7,8-dihydroneopterin on tumor necrosis factor alpha-mediated programmed cell death. We report here that 7,8-dihydroneopterin and hydrogen peroxide upregulate the production of interferon-gamma, thereby establishing an autocrine feed-back loop. Data put emphasis on the role of neopterin-derivates within the cytokine network.

Selective induction of mononuclear phagocytes to produce neopterin by interferons.

Interferon-gamma (IFN-gamma) has been shown to be a potent inducer of neopterin secretion by human peripheral blood monocytes/macrophages (1). In this paper, it is shown that other known stimuli of monocytes (e.g., to secrete proteases or to migrate) such as zymosan-activated human serum, lipopolysaccharide, human C3/iC3 and zymosan coated with complement were unable to trigger monocytes/macrophages to release neopterin. Monocytes/macrophages could be stimulated solely by IFN-gamma (25 U/ml) and IFN-alpha at very high concentrations (10,000 U/ml). In the case of human peripheral blood mononuclear cells (PBMNC), basically the same pattern was observed. If however, in the buffer controls PBMNC showed some neopterin release, all stimuli triggered an increase of neopterin secretion: 10,000 U/ml IFN-alpha induced the same amount of secreted neopterin as did 25 U/ml of IFN-gamma. Both caused higher levels of neopterin secretion than ZAS, LPS and C3/iC3. Amongst the supernatants from PBMNC, only those which were obtained from cells activated with IFN-gamma or -alpha stimulated monocytes/macrophages to produce neopterin. Supernatants from lymphocytes activated with zymosan, lipopolysaccharide and interferon did not contain neopterin, nor did the latter induce monocytes/macrophages to generate and secrete neopterin. Antibodies against IFN-gamma inhibited the triggering effect of the supernatants except when generated by IFN-alpha at 10,000 U/ml. These results demonstrate that both interferons, IFN-gamma and IFN-alpha, the latter only at a 400-fold higher concentration, can trigger monocytes/macrophages directly to secrete neopterin. ZAS, LPS and C3/iC3 are weakly effective only on a mixture of lymphocytes and monocytes/macrophages, provided this cell mixture shows already a basic spontaneous neopterin release.(ABSTRACT TRUNCATED AT 250 WORDS)